The Na-K-C1 cotransporter is a key element in the process of NaCl reabsorption across the mammalian renal epithelium in the thick ascending limb of the loop of Henle, where it is the site of action of diuretics such as furosemide. This transporter also plays a fundamental role in net salt transport in most secretory epithelia, and in non-epithelial cells it is thought to be important in the regulation of cell volume or of extracellular potassium. Previous work on this project has demonstrated that the Na-K-C1 cotransporter is a glycosylated membrane protein 150 to 195 kDa in size, depending on tissue and species. The goal of this project is to understand the mechanisms by which transport is regulated by circulating hormones and cell volume. The proposed studies will be greatly facilitated by the probes and techniques that have been developed in the previous grant period. Specifically: 1) It is proposed to isolate from rabbit and mouse kidney the Na-K-C1 cotransport protein and the cDNA which encodes it, and it produce specific monoclonal antibodies. Analysis of structure of the cDNA and the encoded protein will test the hypothesis of distinct protein isoforms, and it will inform as to important structural features for the remainder of the project. 2) Questions regarding the molecular structure and function of the cotransporter will be addressed using monoclonal antibodies for immunopurification of protein, and an appropriate expression system for study of the functional protein coded by the cDNA. 3) To further understand the regional role of the Na-K-C1 cotransporter in renal salt absorption, the distribution of the Na-K-C1 cotransporter in the rabbit and mouse kidney will be examined using immunocytochemical techniques. These studies will also lend insight into the possible role of membrane trafficking in cotransporter regulation. 4) The hypothesis that stimulation of salt reabsorption in the TALH by vasopressin involves direct activation of the Na-K-C1 cotransporter will be tested by incorporation of radiolabelled phosphate into isolated tubule cells, and subsequent immunopurification of the labeled cotransport protein. Additional experiments will investigate the hypothesis that the information that determines apical/basolateral sorting of the Na-K-C1 cotransporter in polarized epithelia is a part of the protein structure, hybrid constructs of the renal and secretory isoforms will be produced and expressed in epithelial cell lines. Cell surface delivery will be examined by immunocytochemical techniques. Further experiments will refine the search in order to identify the "sorting signal" within the cotransporter structure.